The present invention relates to a method for producing a biologically degradable polyhydroxyalkanoate coating, wherein an aqueous dispersion of polyhydroxyalkanoate is applied to the surface to be coated. In particular, the invention relates to the coating of surfaces which form part of a product to be supplied for consumption to a human being or animal and/or surfaces which form part of a product which is not resistant to high temperatures. A suitable example of a product to be coated is a foodstuff, in particular cheese. The invention also relates to objects coated with such a polyhydroxyalkanoate coating, and also to elastomeric, films of polyhydroxyalkanoate and latices thereof. Elastomeric means the polyhydroxyalkanoates behave at room temperature as elastomers, wherein the crystals serve as physical crosslinks providing a minimum elongation at break of 50%.
Poly(3-hydroxyalkanoates) (PHA) are biologically degradable polymers which can be accumulated by microorganisms as sources of carbon and energy (Anderson and Dawes 1990, Microbiol. Rev. 54:450-472). Poly(3-hydroxybutyrate) (PHB) and the copolymer poly(3-hydroxybutyrate)co-valerate (PHB/HV) are the most known and best studied forms of PHA and are classified as polyhydroxyalkanoates of the PHB type. In the past ten years, a large number of new types of PHA have been found. In particular, Pseudomonas strains have proved to be capable of synthesizing PHAs with diverse compositions (Lageveen et al. 1987, Appl. Environ. Microbiol. 54, 2924-2932). These polyhydroxyalkanoates comprise 3-hydroxy acids having a carbon-chain length of C6-C14 and are termed polyhydroxyalkanoates of the PHA type.
In order to be able to use polyhydroxyalkanoates, they must be derived from biomass and separated from contaminating cell parts such as proteins and lipids. Two important working-up strategies have been developed for PHB and PHB/HV. The first method relates to an extraction with a solvent such as methylene chloride, dichloroethane, chloroform or trichlorobenzene (Lafferty et al. 1988, Biotechnology vol. 6b, 136-176). The disadvantage of this method is that the organic solvents used are toxic and environmentally unacceptable. The use of these solvents would not only make the working-up of PHAs expensive but also considerably limit the number of possible applications of PHAS. All the applications in which PHAs come into contact with products to be applied to or consumed by a human being or animal, such as foodstuffs, are ruled out in this case because small residues of the solvent which is harmful to health can, after all, always be present.
In the second working-up method, the bacteria cells containing PHB/HV are broken open by means of a combined physical, chemical and enzymatic procedure. In this way, the PHB/HV granules are released from the biomass and an aqueous dispersion is produced, viz. a latex. The aqueous PHB/HV dispersion is further purified of cell constituents by means of rinsing steps and centrifugation. A dry PHB/HV powder which can be processed further to produce, for example, foils, small bottles, fibres etc. can then be obtained by means of spray drying the dispersion (Holmes 1985, Phys. Technol. 16, 32-36).
With a view to the environmental requirements, great need has arisen for biologically degradable products. This certainly applies to products which comprise a solid support with a coating. Processing of PHA to form such a coating is an interesting field of application. It is known of PHB and the copolymer PHB/HV that they can be processed in various ways to form a coating. In the first place, by applying a PHB solution in an organic solvent such as chloroform to the surface of the coated agent and allowing the solvent to evaporate. It is clear that this is a very unacceptable method for environmental reasons.
In a second approach, use is made of an aqueous dispersion of PHB, PHB remaining behind as a powder on the surface of the agent to be coated after evaporation of the water. The PHB granules do not fuse and films can only be formed by exposing the surface coated with PHB powder to a solvent such as chloroform (Marchessault et al. 1990, in Novel Biodegradable Microbial Polymers) or by heating the surface coated with PHB powder to the melting point of PHB. This process is disclosed in international patent application WO91/13207 of Marchessault and Lepoutre. Marchessault and Lepoutre describe a method of preparing a coated or impregnated paper or other fibre construct, a latex for use in the preparation of paper or other fibre constructs and films and self supporting films. They describe a latex comprising a colloidal suspension in water of essentially non-crystalline particles of a poly-xcex2-hydroxyalkanoate polymer or copolymer and illustrate butyrate and valerate as preferred hydroxyalkanoates. Their latex will comprise 10 to 50%, preferably 15 to 30%, more preferably 20 to 25% by weight of the PHA. Typically their latex will comprise 10 to 15 parts by weight. They describe that a film can be made from a latex by casting a dilute solution of 15-25% w/w solids on an impervious surface and allowing the water to evaporate slowly at room temperature. They state that heating the air dried film in a drying oven at a temperature 30 degrees below Tm causes moderate fusion. These films have a microporous structure and can be hot pressed at 100-140xc2x0 C. and 1000-5000 psi to produce flexible translucent films of average to high crystallinity. The hot fusion treating of PHB/V copolymer containing 21% by weight PHV provides water imperviousness. Dense transparent films are formed by exposing air dried films to solvent or liquid vapour atmospheres such as chloroform and other halogenated solvents. Explicitly Marchessault and Lepoutre describe xe2x80x9cA few grams of 21% HV polymer latex was diluted to 20-30% w/w solids with distilled water. The resulting mixture was poured on a clean glass surface and allowed to air dry at room temperature overnight. A white uniform coat or film with little or no strength was obtained. It was then placed in a convection oven set at 100xc2x0 C. for a period of not less than 10 min. A partially fused film was obtainedxe2x80x9d. Hot pressing the resulting film provided a film with at most elongation at break of 30%. Alternatively subjecting the partially fused film to chloroform provided an extremely smooth and tough film.
Disadvantages also apply to these known methods, firstly the use of an organic solvent and secondly, the performance of a heating step in which a temperature above 100xc2x0 C. has to be used. The applications are therefore also clearly limited in this case. Marchessault and Lepoutre""s main application is for impregnating paper. They disclose nothing with regard to preparing elastomeric films. Their coatings are either microporous and non-peelable or thermoplastic exhibiting at most elongation at break of 30%. They do not disclose elastomeric films suitable for appliance to or consumption by humans or animals.
DE-4 040 158 describes how the use of organic solvents is harmful to health and how the presence of the solvent in the coating makes it impossible to use them in the foodstuffs or pharmaceutical industry, in particular as packing material also for material for treating wounds. A further disadvantage mentioned is that the known coatings are available solely as impermeable films. For many applications, however, such as medical bandaging or other materials for absorbing body fluids, a porous coating, in particular a coating permeable to gas and air, is necessary, which materials cannot be obtained with the aid of the coatings with solvents. In DE 4 040 158 a dispersion of polyhydroxybutyrate or polyhydroxyvalerate, or copolymers thereof, in water is described for obtaining such a porous coating. The aqueous dispersions can be prepared by stirring PHA powders or directly in the processing of PHA-forming organisms. The coated objects are prepared, according to DE 4 040 158, by applying the dispersion of PHA to the object and then subsequently drying to remove the dispersing agent. Depending on the drying conditions, a more or less porous coating or an impermeable and continuous coating film can be obtained. The degree of porosity can be controlled by the choice of drying conditions and the granule size of the polymer particle. If a continuous and impermeable coating film is desired, heating is carried out to temperatures at which the coating melts to form a film either during the drying or subsequently to the drying. According to DE 4 040 158, a porous non-film-forming coating is advantageous where gas and air permeability is necessary or desirable, as in the case of medicinal bandaging for wound treatment. A porous coating is obtained by not heating the PHA coating to the melting point so that a film is not formed. In certain cases, sintering in the region of the melting point is advantageous to produce coatings having a defined porosity. The melting point is 173.3xc2x0 C. for polyhydroxybutyrate and it is 126.6xc2x0 C. for polyhydroxybutyrate-co-hydroxyvalerate, with a ratio of 80:20 mol %. As examples of surfaces to be coated, mention is made of nonwoven fabric, woven fabric or plastic foil, cellulose, wood, textile fibres, metal or glass.
Various products, in particular food products such as cheese, are coated with a synthetic, non-biologically degradable plastic. The plastic of this rind cannot be recovered or be recycled and presents a problem for the environment. Furthermore, such a coating must fulfil a large number of functions and a large number of requirements. The coatings which have been described above do not fulfil the requirements which are imposed on a cheese coating or a coating for a temperature-sensitive agent. The coating material serves to prevent mechanical damage and also attack by moulds. The coating must offer both mechanical and hygienic protection. Furthermore, the coating must preferably be easy to apply, for example by immersion or coating with a sponge or brush. In particular, in the case of cheese, the coating advantageously affects the lustre and rind formation and it must adhere well to the wet surface of the cheese. In addition, after drying, a coating must be produced which does not crack and does not stick, for example, to boards or other cheeses. The coating should also be semipermeable since CO2 and certain other flavouring components must be able to diffuse through the coating in order not to adversely affect maturing. Water vapour must also be allowed to pass through in a controlled manner so that natural maturing can occur without any question of excessive weight loss. On the other hand, components must not diffuse into the cheese from the coating. Furthermore, the coating must keep well during prolonged storage, there must be good adhesion to any paraffin layer to be applied and there must be no peeling after vacuum packaging. In addition, the machines which are used must be easy to clean. It will be clear that these properties apply not only to cheese coatings but are generally desirable.
Although biologically degradable and compostable coatings exist, they are generally water-soluble polymers which are consequently also water-sensitive. Examples of these are polyvinyl alcohol, polyethylene oxide, and also proteins such as gelatine. Hitherto, there has been no possible use of polyhydroxyalkanoate films as a coating in the foodstuffs industry, on the one hand, because of the use of organic solvents and, on the other hand, because of the need to heat the polyhydroxyalkanoates to the melting point, which could unnecessarily damage the product.
The object of the present invention is to solve the problems explained above. The present invention relates therefore to a method for producing a biologically degradable poly(3-hydroxyalkanoate) coating in the form of an elastomeric film, in which an aqueous dispersion of poly(3-hydroxyalkanoate) is prepared and the dispersion is applied to the surface to be coated, after which water is made or allowed to evaporate to obtain a film, the film formation taking place at a temperature lower than the melting point of the polyhydroxyalkanoate, and the polyhydroxyalkanoate used being a polymer which is made up of saturated or unsaturated 3-hydroxy fatty acids having a carbon-chain length of 6-14. The 3-hydroxy fatty acids can therefore contain 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms suitably the method can even be carried out at a temperature more than 35xc2x0 C. below the melting point.
A method for producing a biologically degradable polyhydroxyalkanoate coating in the form of an elastomeric film, in which an aqueous dispersion of polyhydroxyalkanoate is prepared and the dispersion is applied to the surface to be coated after which the water is made or allowed to evaporate to obtain a film, the film formation taking place at a temperature lower than the melting point of the polyhydroxyalkanoate with a polymer which occurs in Pseudomonas being used as polyhydroxyalkanoate, with the proviso the polyhydroxyalkanoate is not a polymer or copolymer of xcex2-hydroxybutyrate or xcex2-hydroxyvalerate, also forms part of the invention. The method for producing coating in the form of an elastomeric film according to the invention does not require further steps of hot fusion, hot pressing or contact with chloroform or halogenated solvent to obtain the elastomeric characteristic. Selection of specific hydroxyalkanoates as starting material in an aqueous dispersion ensures elastomeric properties are acquired merely upon drying the aqueous dispersion after applying it to the surface to be coated. On the basis of what was known regarding film formation and coatings of polyhydroxybutyrate and polyhydroxyvalerate and copolymers thereof it was totally unexpected that films having an elastomeric character could so simply be obtained. The films according to the state of the art with PHB and PHV required additional measures as described in detail above. Furthermore it could not be predicted that hydroxyalkanoates made up of saturated or unsaturated 3-hydroxy fatty acids having a carbon chain length of 6-14 would be suitable for preparing an aqueous dispersion even having characteristics suitable for use as a coating at all. Such an aqueous dispersion could be expected to have a chewing gum like structure in water or hydrophilic media. It also was not to be expected what the properties of any resulting film would be and certainly not that they would be so fine or so differnt from those of the described PHB and PHV films. Suitably the method according to the invention can be carried out at a temperature more than 35xc2x0 C. lower than the melting point of the polyhydroxyalkanoate. Preferably, a polymer is used which occurs in fluorescent Pseudomonas belonging to the RNA homology group I. The polymers of PHA occurring in fluorescent Pseudomonas belonging to the RNA homology group I do not, after all, comprise PHB or PHB/HV and have such low melting points, glass transition temperatures and crystallinity that the film formation takes place at a temperature lower than the melting point of the polyhydroxyalkanoates and can even occur at a temperature more than 35xc2x0 C. below the melting temperature.
PHB and the copolymer PHB/HV, the polyhydroxyalkanoates of the PHB type, are typically thermoplastics having a high melting point (120-180xc2x0 C.) (Byrom 1987, TIBTECH 5, 246-250). These polymers are highly crystalline. PHAs formed by Pseudomonades, in particular by fluorescent Pseudomonades belonging to the RNA homology group I have, on the other hand, typically elastomeric properties. The melting point of this category of polyhydroxyalkanoates varies between 40 and 60xc2x0 C. and the crystallinity depends on the composition, but is between 0 and 30% (Marchessault et al. 1990, Ind. J. Biol. Macromol. 12, 158-165). Pseudomonas strains can easily be brought in a known manner to high production levels of PHA (Eggink et al. 1992, Proceedings of ACS Division of Polymeric Material 67, 130-131). Pseudomonas produces no PHB, and therefore, the polyhydroxyalkanoates which are formed by Pseudomonas can be used in an outstanding way in a method according to the present invention without first extracting PHB therefrom. In particular. because the working up of polyhydroxyalkanoates from microorganisms provides an aqueous dispersion, mixtures of polyhydroxyalkanoates occurring in microorganisms, in particular, are extremely suitable for use in a method according to the present invention. The working-up and coating can be performed continuously and in a minimum number of steps. As already stated, the PRA-containing bacteria cells can be broken open by means of a combined physical, chemical and enzymatic procedure, as a result of which the PHA granules are released from the biomass and an aqueous dispersion is produced which can be purified further from contaminating constituents by means of washing steps and centrifugation. With the present method, spray drying of the dispersion and adding of organic solvents are no longer necessary. The processing of PHA to form a coating can be (carried out) just by applying the dispersion as it is obtainable directly by working-up from the microorganisms and by allowing it to dry. The polyhydroxyalkanoates of the PHB type probably do not form an impermeable film because of the high crystallinity and the high glass transition temperature of 0-4xc2x0 C. the invention in all embodiments (method of preparation, film, coated product, latices) is preferably directed at polyhydroxyalkanoates having a glass transition temperature below 0xc2x0 C.
In the method according to the invention, a high-temperature step is unnecessary for sintering or melting for the purpose of film formation, in contrast to the method which was known for poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate)-co-valerate coatings. Neither the presence of an organic solvent nor an intermediate step in which the aqueous dispersion of PHA is dried to form a powder and then dissolved in an organic solvent is necessary. With the method according to the present invention, it is therefore possible to coat products which may not be exposed to an excessively high temperature and/or to coat products which are applied to or consumed by a human being or animal and in which organic solvents must not be present. The coating does not per se need, however, to be applied to or consumed by the animal or the human being.
The method according to the present invention can be performed at a temperature in the range of 0-30xc2x0 C., preferably at a temperature in the range of 10-18xc2x0 C. The choice of temperature employed will depend on the product to be coated and on economic considerations. The PHA or PHAs used will also be a factor on which the temperature to be used depends.
The method according to the invention can be performed with good results using a dispersion whose dry-matter content of PHA is at least 10%. Outstanding results are achieved with a dry-matter content higher than 30% and even higher than 50%. Dispersions with a dry matter content higher than 65% have been shown to be suitable embodiments for use in preparing elastomeric films according to the invention.
The method in which an aqueous dispersion according to the present invention for coating a product is used has been performed by the inventors with PHAs of the PHB type such as PHB/HV and PHB but these did not yield a film, only a powdered coating whose powder granules exhibited no or little mutual adhesion. The known coating could not be peeled off as a film from the coated product. The coating did not have the mechanical strength of a film. The inventors results agree with what has already been stated in DE 4 040 158 and PCT 91/13207 for PHB and the copolymer of PHB with hydroxyvalerate. A film having good mechanical strength and containing PHB granules can only be formed by exposing the surface coated with PHB powder or aqueous dispersion to a solvent such as chloroform or by heating to the melting point.
Polyhydroxyalkanoates of the PHA type with saturated or unsaturated 3-hydroxy fatty acids having a carbon-chain length between C6 and C14 are, inter alia, produced by Pseudomonas strains, in particular fluorescent Pseudomonas strains belonging to the RNA homology group I. This category of hydroxy fatty acids has typically elastomeric properties, with a melting point between 40 and 60xc2x0 C. and a crystallinity between 0 and 30% (Marchessault et al. 1990, Ind. J. Biol. Macromol. 12, 158-165) and yields outstanding films by the present method. Preferably, the method according to the invention is performed with polyhydroxyalkanoate copolymers which comprise at least 3-hydroxy acids having carbon-chain lengths of C8-C10. Outstanding results are obtained if the mixture comprises at least 50% C8-C10 PHAs.
An advantage of the use of dispersions is that standard additives such as stabilizers, plasticizers, fillers, dyes, emulsifiers. thickening agents, antioxidants, antistatics, preservatives, crosslinking agents, etc. can be added to the dispersion and the properties of the film can be simply improved and modified thereby. In addition, the presence of the water phase offers the possibility of mixing the PHA with other water-soluble, biologically degradable polymers. Other biologically degradable polymers which can be taken up in the dispersion comprise polyvinyl alcohol, gelatine, methylcellulose, polyethene oxide, pvp, starch. Furthermore, an anti-fungal agent can be taken up in the dispersion.
As stated above, the method according to the present invention can advantageously be used to coat foodstuffs, in particular for coating cheese. The film which is obtained by the present method is outstandingly suitable for coating cheese. All the requirements which are imposed thereon with respect to a suitable cheese coating are fulfilled by the coating which can be obtained by the present invention. In particular, the mechanical strength of the coating is a positive factor. The fact that the PHA granules yield, without heating to the melting point, a film having sufficient mechanical strength to function as cheese coating is very surprising since PHAs of the PHB type yielded only a powdered coating. The film obtained resembles a xe2x80x9clatexxe2x80x9d film and is easy to distinguish from a coating which is obtained with an aqueous dispersion of the PHB type without the heating step to the melting point of the PHA. In an embodiment which is particularly suitable for producing a coating or film according to the invention, a drying time of less than 24 hours at an atmospheric humidity of 85% and a temperature of 10-18xc2x0 C. may be adequate. This method according to the invention is outstandingly suitable for producing a cheese coating.
The bacteria cells of Pseudomonas can readily be cultured, the polyhydroxyalkanoates formed depending on the medium on which the Pseudomonas is cultured. Thus, various fatty acids, such as oleic acid and linoleic acid, can be used as hydroxy fatty acids for the PHA to be formed.
Polyhydroxyalkanoates of other microorganisms may, of course, also be used in the method according to the present invention, provided they are saturated or unsaturated 3-hydroxy fatty acids having a carbon-chain length of 6-14. The composition of the polyhydroxyalkanoates may be controlled by choosing the media on which the microorganisms are cultured so that, on the one hand, the presence of PHB and PHB/HV can be avoided in the polyhydroxyalkanoate mixture to be used for the method and, on the other hand, the composition of the polyhydroxyalkanoates produced by a microorganism and, consequently, also the properties of the film can be precisely controlled.
The present invention also relates to a product coated with a polyhydroxyalkanoate film, said film being elastomeric and said film comprising polyhydroxyalkanoate made up of saturated or unsaturated 3-hydroxy fatty acids having a carbon-chain length of 6-14. The invention also comprises a product coated with a polyhydroxyalkanoate film, said film being elastomeric and said film comprising polyhydroxyalkanoate which occurs in Pseudomonas, preferably polyhydroxyalkanoate which occurs in fluorescent Pseudomonas belonging to the RNA homology group I with the proviso the polyhydroxyalkanoate is not a polymer or copolymer of xcex2-hydroxybutyrate or xcex2-hydroxyvalerate. In particular a coated product according to the invention can be a product to be applied to or consumed, by a human being or animal or a product which must not be exposed to 100xc2x0 C. or more. A suitable product is cheese. The product coated with polyhydroxyalkanoate film may also be composed of a mixture of polyhydroxyalkanoates of the abovementioned type. The invention relates to all products coated with polyhydroxyalkanoate film of the above type which can be prepared by the method according to the invention. The products coated with a polyhydroxyalkanoate according to the invention do not consist of poly(3-hydroxybutyrate) or a copolymer thereof, poly(3-hydroxybutyrate)-co-valerate or poly(3-hydroxyvalerate) since, to prepare such coatings, a heating step is necessary to form a film. Besides this these polymers or copolymers do not provide elastomeric films as is clear from the literature cited above.
The invention furthermore comprises an elastomeric film of polyhydroxyalkanoate in which the polyhydroxyalkanoate is made up of saturated or unsaturated 3-hydroxy fatty acids having a carbon-chain length of 6-14. The invention also comprises an elastomeric film of polyhydroxyalkanoate in which the polyhydroxyalkanoate occurs in Pseudomonas, preferably in fluorescent Pseudomonas belonging to the RNA homology group I with the proviso the polyhydroxyalkanoate is not a polymer or copolymer of xcex2-hydroxybutyrate or xcex2-hydroxyvalerate. Preferably a film according to the invention is water resistant. I n particular, the invention relates to an elastomeric film of polyhydroxyalkanoate which is suitable as a coating for products suitable to be applied to or consumed by human beings and/or animals. The film according to the invention can suitably be applied to hydrophilic surfaces and will exhibit good attachment properties to such a surface without requiring a heating step or any other step than drying at a temperature below Tm. In fact temperatures more than 35xc2x0 C. below Tm can even be applied. Cheese is a suitable product. A preferred film according to the invention will comprise an elongation at break of at least 50%. Suitably the elongation at break will be over 200%. The dry-matter content of a film according to the invention can be more than 10%. Preferably it is higher than 30%, even higher than 50%. Another property of a film which is suitable in particular as cheese coating is a film which contains less than 4.5% of parts which are soluble in water. The typical gas permeability is 3-5 l/m2/hr at a temperature of 20xc2x0 C. and relative humidity of 90-0% for a film according to the invention. These values being for a film with a thickness of 0.1 mm. Not only is the invention directed at the novel and inventive films but also at latices useful for production of such films as can be produced in the various embodiments of the preparation method of coated products according to the invention.
Thus an aqueous latex-like dispersion of polyhydroxyalkanoate made up of saturated or unsaturated 3-hydroxy fatty acids having a carbon chain length of 6-14 falls within the scope of the invention. Also an aqueous latex-like dispersion of polyhydroxyalkanoate, wherein the polyhydroxyalkanoate occurs in Pseudomonas, preferably polyhydroxyalkanoate which occurs in fluorescent Pseudomonas belonging to the RNA homology group I, with the proviso the polyhydroxyalkanoate is not a polymer or copolymer of xcex2-hydroxybutyrate or xcex2-hydroxyvalerate falls within the scope of protection. Such dispersions preferably having a pH higher than 8, preferably higher than 10. Suitably the pH is 12 as illustrated in the Examples. A dispersion according to the invention may further comprise more than 30%, preferably more than 50%, in particular more than 65% polyhydroxyalkanoate as dry weight. Preferably an aqueous latex-like dispersion according to any of the aforementioned embodiments is substantially free of compounds toxic to a human being or animal. In addition it is substantially free of contaminant cell rests of cells from which the polyhydroxyalkanoate has been derived.